Most flowering plants have undergone genome duplications during their evolution. Sequencing plant genomes has revealed that most eudicot genomes descended from an ancient hexaploid ancestor and then underwent lineage-specific whole genome duplication (polyploidization). Although this polyploidization has not occurred in grape
, it has occurred once in poplar
[4, 5], and potato
, and twice in Arabidopsis and Brassica, the latter of which has undergone additional whole genome triplication (WGT) since its divergence from the Arabidopsis lineage
[8, 9]. Polyploidy results in the accumulation of homologous genes in the genome leading to increased complexity and redundancy. Redundant copies of amplified genes in the duplicated genome can diverge soon after duplication, and they might become pseudogenes (nonfunction), or gain additional or novel functions (subfunction and neofunction, respectively). In addition, duplicated genes may play a role in preventing potential harmful mutations (buffering). Regardless of their alterations, the ultimate fate of duplicated genes vary according to the individual plant and species
[10, 11]. Recent studies of several species have demonstrated that a broad range of genetic and epigenetic responses also occurred soon after polyploidization, including DNA deletions, chromosome rearrangements, cytosine methylation, gene silencing, the activation of transposons, and the modification of parental imprinting
[12–15]. These events have been associated with small RNAs
, indicating that changes in the small RNAs of polyploidy genomes provide insight into the control of the genetic and epigenetic mechanisms that occur in response to genome duplication.
Small RNAs are short noncoding RNAs typically 19–25 nucleotides (nt) in length with two prominent sizes of 21 and 24 nt. In general, microRNAs (miRNAs) correspond to the 21 nt class of small RNAs and short-interfering RNAs (siRNAs) comprise the 24 nt class. Small RNAs have been shown to be involved in a broad range of functions including heterochromatin formation, DNA methylation, and gene silencing
[17, 18]. By targeting genes for cleavage or repressing protein translation, plant miRNAs play an essential role in various biological and metabolic processes, including tissue identification, developmental control, and response to environmental stress. In contrast, siRNAs seem to function as guardians against transposable elements during plant development
[12, 19–22]. While both classes of small RNAs have been characterized, recent studies have focused mainly on miRNAs because they regulate diverse developmental processes through the direct cleavage of target messenger RNA (mRNA). The biogenesis of miRNA takes place in a stepwise manner. miRNA is initially transcribed by RNA polymerase II. A long primary transcript (pri-miRNA), which forms a hairpin-like secondary structure, is then processed by the nuclear localized Dicer-like 1 (DCL1) in the plant nucleus to release a 60~70 nt intermediate. The intermediate, known as precursor miRNA (pre-miRNA), has a hairpin structure with base-pairing that is not perfectly complementary, resulting in many mismatches and bulges that are further processed into a miRNA/miRNA* duplex. After undergoing 3’-methylation, a paired set of miRNAs are then exported from the nucleus to the cytoplasm by HASTY
. Subsequently, the mature miRNA is loaded onto the RNA-induced silencing complex (RISC) and guides the RISC to recognize complementary sites on target mRNAs, resulting in transcript cleavage
[22–24] or translational repression
Many miRNAs have been identified by computational or experimental approaches in various plants. Genome-wide analyses of miRNA have revealed that several miRNA families are highly conserved among plant genomes (conserved miRNAs), though individual species also possess highly specific and evolved (or evolving) miRNAs genes (novel miRNAs). It has been reported that conserved miRNA families have been expanded by duplication followed by subsequent reduction of redundant homologs whereas novel miRNAs, which are often expressed only in restricted species, might initially evolve neutrally but develop more specialized roles
[27–32]. Sequence similarity-based approaches can be used to identify conserved miRNA candidates from newly-sequenced genomes. However, due to their species-specific nature, it is difficult to identify novel miRNAs by computational or hybridization-based methodologies. Recent advances in high-throughput sequencing technologies have facilitated the discovery of both conserved miRNAs and the less abundant novel and non-conserved miRNAs in plants
[30, 33–36]. Despite their importance in plant development, the genomic origin and evolution of miRNAs in polyploid genomes have not been well-described.
Brassica rapa, one of the two ancestral species of oilseed rape, is a member of the Brassicaceae family, which includes the model plant Arabidopsis thaliana and over 300 other genera. B. rapa shows great morphological plasticity, which has led to its domestication and selective breeding into a range of different crop types, such as Kimchi cabbage (Korean heading form), turnip, Mizuna, and rape mustard. This morphological diversity makes B. rapa an excellent species for the study of plant morphology evolution as well as the process of domestication and directed selection. An annotated draft genome sequence for Brassica rapa ssp. pekinensis cv. Chiifu was recently reported by the multinational Brassica rapa genome sequencing project consortium
. Comparative genomic analyses showed that the B. rapa genome had extensive collinearity with the genome of A. thaliana, evident in its triplicated and rearranged genome blocks. The extent of gene loss (fractionation) varied between the related genome segments of B. rapa, with one copy containing a greater proportion of genes expected to have been present in its ancestor (70%) than the remaining two (36% and 46%)
. A rapid evolutionary rate and the specific copy number amplifications of particular gene families are believed to contribute to the remarkable morphological plasticity of Brassica species. Therefore, the B. rapa genome sequence provides an important resource for studying the evolution of polyploidy genomes and the epigenetic regulation of duplicate genes.
To study epigenetic control in a triplicated crop genome, the genome-wide identification of B. rapa miRNAs is highly essential. Recent studies have identified a limited number of small RNAs in B. rapa. Most of these sequences were conserved miRNAs based on computational similarity searches with restricted sequence datasets, with very few novel miRNAs identified
[38–40]. The main objective of this study was to identify putative miRNA candidates of B. rapa using a draft of its whole genome sequence. We identified miRNAs based on small RNA deep sequencing along with similarity searches, and then characterized the genomic organization and evolutionary origin of the miRNAs. In addition, we predicted the target genes of the candidate miRNAs and analyzed the putative function of miRNA targets. We constructed a web interface for the public to access the data generated in this study in order to provide a resource for studying the epigenetic control of gene expression in Brassica species.